小鼠再生肝胞浆磷酸酪氨酸蛋白磷酸酶的纯化和性质研究

Purification and Properties of Cytosolic Phosphotyrosyl Proteinphosphatase From Regenerating Liver

以^(32)P(Tyr)-Poly Glu,Tyr(4:1)为底物,用于研究小鼠再生肝胞浆磷酸酪氨酸蛋白磷酸酶(PTPP)的分离纯化和性质。再生肝胞浆经60％饱和度硫酸铵盐析,二次DEAE纤维素层析,Sephadex G-200柱层析和Poly Glu,Tyr-Sepha-rose 4B亲和层析后,得到的PTPP分子量为67000,纯度提高1123倍,活性回收率为28％,对^(32)P(Tyr)-Poly Glu,Tyr有很高的活力,对^(32)P(Ser/Thr)-Casein(酪蛋白)和PNPP(对硝基苯酚磷酸盐)没有作用,其最适pH为6.8～7.1,对热不稳定。EDTA对酶有激活作用,Zn^(2+)、PNPP、P-Tyr、多胺化合物、焦磷酸根、钼酸根、柠檬酸根对酶有明显的抑制作用。酶对Na_3VO_4不敏感。碱性蛋白质组蛋白、鱼精蛋白对酶活力有抑制作用,酸性蛋白质酪蛋白和酸性多糖物质肝素对酶活力有激活作用,且后者能减弱前者的抑制作用。

Using a peptide poly (Glu,Tyr)(4:1) phosphorylated at tyrosine residue by protein-tyrosine kinase from rat spleen as substrate, purification and properties of phosphotyrosyl protein phosphatase (PTPP) from regenerating mouse liver cytosol was studied.Most of the PTPP activity in extract after (NH4)2 SO4 fraction of regenerating liver cytosol were absorbed on DEAE cellulose (DE52).The eluted PTPP indicated the presence of at least five species.The main PTPP activity was further purified by chromatography on DE52,Sephadex G-200 and poly (GIu,Tyr) (4:1)-Sepharose 4B.Overall purification of about 1123-fold was achieved with 28% recovery of enzyme activity.The properties of this purified PTPP was follows, (1) The enzyme showed only one band of molecular weight about 67KDa by polyacrylamide pore gradient and SDS-gel electrophoresis;(2) It exhibited high specificity towards 32p(Tyr)poly(Glu, Tyr)(4:1), but did not hydrolysed 32p(Ser/Thr) casein and PNPP, (3) Its optimum PH was 6.8-7.1 and it was unstable to heat treatment; (4) Its activity was activated by EDTA, but not activited by Mg2+, Mn2+, Ca2+, (5) Its activity was inhibited by Zn2+, Co2+, PPi, PNPP, P-Tyr, MoO4 and citrate,but not inhibited by F-,Ac-, HPO42-, and VO43-.Low molecular weight nucleotides ATP, ADP, AMP were inhibitors.(6) Its activity was inhibited by histone and protamine, but stimulated by casein and heparin.